E. coli and integrated them into a low-cost microfluidic sensor to measure the conductometric transduction of CIP–bacteria binding events. The sensor comprised two CIP-MWs suspended perpendicularly to a PDMS microchannel. The inter-wire electrical resistance of the microchannel was measured before, during, and after exposure of CIP-MWs to bacteria. A decline in the inter-wire resistance of the sensor after 30 min of incubation with bacteria was detected. Resistance change normalization and the subsequent analysis of the sensor’s dose-response curve between 0 to 109 CFU/mL bacteria revealed the limits of detection and quantification of 2.1 × 105 CFU/mL and 7.3 × 105 CFU/mL, respectively. The dynamic range of the sensor was 104 to 107 CFU/mL where the bacteria counts were statistically distinguishable from each other. A linear fit in this range resulted in a sensitivity of 7.35 μS per CFU/mL. Experiments using competing Sarcina or Listeria cells showed specificity of the sensor towards the imprinted E. coli cells. The reported CIP-MW-based conductometric microfluidic sensor can provide a cost-effective, durable, portable, and real-time solution for the detection of pathogens in water.The rapid, inexpensive, and on-site detection of bacterial contaminants using highly sensitive and specific microfluidic sensors is attracting substantial attention in water quality monitoring applications. Cell-imprinted polymers (CIPs) have emerged as robust, cost-effective, and versatile recognition materials with selective binding sites for capturing whole bacteria. However, electrochemical transduction of the binding event to a measurable signal within a microfluidic device to develop easy-to-use, compact, portable, durable, and affordable sensors remains a challenge. For this paper, we employed CIP-functionalized microwires (CIP-MWs) with an affinity towards  There has been an increasing demand for the inexpensive, rapid, and portable monitoring of pathogenic and indicator bacteria in medical and environmental samples ,2. The cPoC devices use various transduction techniques such as electrochemiluminescence ,7, surfaMolecularly imprinted polymers (MIPs), also called “synthetic antibodies”, are polymeric recognition materials with a robust and competitive affinity to target molecules like their bio-recognition counterparts and the added value of polymers’ stability in harsh environments ,17. MIPsE. coli with high efficiency. As a versatile group of compounds, MIPs can recognize analytes of many sizes, shapes, and chemical functionalities, like proteins, membrane glycolipids, and even whole bacteria cells ,21,22,23Electrochemical sensors have been successfully integrated with MIPs and used with various readout methods including field-effect transistors, conductometry, electrochemical impedance spectroscopy (EIS), voltammetry, and amperometry. These platforms convert the interaction between MIPs and target molecules on an electrode surface into various electrical signals . ConductE. coli bacteria into a quantitative electrical signal. By monitoring the resistance of bacteria suspensions with various cell counts in a microchannel between two suspended CIP-MWs, the effect of captured cells by the CIP coatings on the conductance signal was studied. The developed sensing platform can be integrated into a hand-held device in the future, enabling the on-field and low-cost monitoring of pathogens.Microfluidics has enabled the fabrication of field-deployable and low-cost sensors, which would need a small sample volume and a short time for biodetection . VariousE. coli OP50, Sarcina lutea (#155420), and Listeria innocua (#33090) were obtained from the Caenorhabditis Genetics Center , Carolina Biological Supply Company, , and American Type Culture Collection , respectively. All chemicals including acetonitrile, methanol, sulfuric acid, acetic acid, tetraethoxysilane, phosphate-buffered saline (PBS), sodium chloride (NaCl), 2, 2′-azoisobutyronitrile (AIBN), ethylene glycol dimethacrylate (EGDMA), methylmethacrylate (MMA), N-vinylpyrrolidone (VP), acrylamide (AAM), methacrylic acid (MAA), Luria Broth (LB), and LB Broth with agar were purchased from Sigma-Aldrich . Stainless steel microwires  were used as CIP coating substrates and electrodes in the microfluidic device. Bacteria strains including g for 15 min. The pellet was resuspended into fresh PBS (pH 7.0). Plate culturing and colony counting were used to determine the bacteria count when needed [All the bacterial strains were cultured in LB liquid growth medium overnight inside a shaker incubator at 37 °C and 150 rpm. The supernatant was removed after centrifugation at 7000× n needed ,41. 9 CFU/mL in fresh PBS was centrifuged at 7000× g for 15 min, and the pellet was resuspended in the 3 ppm NaCl solution. This process was repeated three times to remove excess PBS. Serial dilution of the bacteria suspension in 3 ppm NaCl solution was performed to obtain the lower bacteria counts for dose–response investigations of the sensor. Trypan blue staining of bacteria exposed to 3 ppm NaCl electrolyte for 30 min indicated that the cells were viable after the exposure. For obtaining the bacteria sample for sensor characterization, 3 ppm NaCl in deionized water was used as a non-fatal  carrier E. coli cells and fluorescent microscopy were used. The mean green intensity of the images was measured with RGB measure plus plugin of Image J software (version 1.54e) and used for comparing the fluorescent intensity.To examine bacteria capturing by functionalized MWs within the microfluidic device, green fluorescent protein (GFP)-tagged MIPs have been synthesized on various flexible metals as supporting substrates including gold, silver, platinum, copper, titanium, and stainless steel (SS). Among these, SS has gained great interest due to its low cost, non-toxicity, corrosion resistance, durability, and rigidity . FurtherTM, Thermo Fisher Scientific, Dreieich, Germany) for 30 min, when the solution color changed from clear to white. An E. coli OP50 suspension  was centrifuged at 3000× g for 5 min, and the supernatant was removed. The pellet was resuspended into 1.5 mL of pre-polymer solution transferred into a centrifuge tube. The surface-functionalized SS-MWs were then immersed in the tube, and the tube was sealed. Polymerization was performed at 65 °C for 11 h. The first 30 min was performed while tubes were rotating using a tube rotator inside the oven to increase the homogeneity of CIP coating along the surface of the MW. CIP pre-polymers were prepared by dissolving MAA (180 µL), AAM (21 mg), VP (4.2 µL), MMA (5.2 µL), EGDMA (570 µL), and AIBN (30 mg) in acetonitrile (2.2 mL). These quantities were optimized previously in a design of experiment exercise, leading to form a uniform and stable CIP coating with a thickness of 2.2 ± 0.4 μm on MWs . The solv/v: 9/1), methanol, and DI water for 20 s to remove bacteria templates from the CIP coatings and generate associated cavities for rebinding. As a control group, non-imprinted polymer (NIP) MWs were prepared simultaneously using an identical synthesis procedure, without adding E. coli templates. After polymerization, the CIP-MWs were washed with methanol-acetic acid  layers containing the design of a microchannel (500 μm × 900 μm), the network of inlet–outlet, and two MW channels (130 μm ×130 μm) with an inter-wire distance of 1.5 mm A. Each l0) was used as the baseline resistance of the sensor. Next, the bacteria suspension at a known concentration was run through the microchannel for 30 min to allow incubation and conjugation of bacteria with CIP cavities. Subsequently, the second electrical resistance measurement, R1, was performed. The last resistance measurement, R2, was performed after running a blank electrolyte for the second time (post-incubation electrolyte 2 wash) through the microchannel for 10 min. Measurements were repeated five times at each step and for three replicate devices.The electrical resistance measurement was performed in three stages. In the first stage, referred to as pre-incubation electrolyte 1 wash, 3 ppm NaCl solution as a blank electrolyte was infused through the microchannel, and the measurement was performed. The resistance value obtained at this stage  to eliminate the variability among devices. The sensor dose–response curve was generated based on the normalized resistance difference method (ΔR/R0 = (R0 − R2)/R0). Here, R1 was not used as this resistance includes the effect of bacteria attached to the wire but not necessarily captured by the CIP cavities. Experiments at various bacterial counts were performed and the sensor dose–response curve of ΔR/R0 versus bacteria count was plotted. The obtained RMinitab 16 statistical software was used for statistical data analysis. A significance level of 0.05 was adopted. The Mann–Whitney U test, also known as the Wilcoxon rank sum test as the nonparametric version of the parametric t-test, was utilized to identify if any two pairs of means were significantly different . p-value < 0.05). The linearity of the sensor was determined by line fitting into the dynamic range of the dose–response curve and analyzing the R2 values. The limit of detection (LOD) and limit of quantification (LOQ) were determined as bacteria counts with ΔR/R0 equal to that of the blank NaCl solution plus 3 and 10 times its standard deviation (SD), respectively. The sensitivity of the sensor in the linear range of the dose–response curve was found based on the slope of the linear range response. The dynamic range of the sensor was identified based on the statistical difference of normalized resistance responses between adjacent bacteria counts -tagged When uncoated SS-MWs were installed in the microfluidic device , top rowWhen a microfluidic device with a pair of NIP-MW electrodes was used , bacteria incubation at 106 CFU/mL (R1), and post-incubation wash (R2) are presented in After observing the bacteria-capturing characteristics of the SS-MWs, NIP-MWs, and CIP-MWs, we aimed to understand the response time of our sensor in terms of reading a constant electrical resistance upon applying a sweep current in the range of 10 nA–1 μA with a step size of 10 nA. Accordingly, the timelapse resistances of an SS-MW-based microfluidic sensor at three stages of pre-incubation wash  than the baseline resistance. This decrease can be attributed to the presence of suspended bacteria cells between SS-MWs within the microchannel that facilitate the charge transfer between the electrodes. However, the resistance increased to the baseline resistance (R2/R0~1) after the post-incubation wash, confirming that all the bacteria cells were washed away with the second electrolyte, expectedly.Repeat experiments for three SS-MW-based sensors were performed, and the average normalized resistances before, during, and after bacteria incubation are plotted in 2/R0 < 1, p-value = 0.012). This decrease in the resistance after washing the bacteria from the microchannel could have been due to the non-specific absorption and attachment of bacteria to the surface of NIP-MWs, which is demonstrated in p-value = 0.0008) than the NIP-MWs. The increased normalized resistance drop resulted from the strong attachment of bacteria cells to the CIP-MW cavities that could not be washed away during the post-incubation wash. The results in E. coli bacteria suspensions with cell counts between 0 and 109 CFU/mL were prepared with serial dilution and used in these experiments. 2/R0) of the CIP-MW-based sensor along with the responses obtained from two parallel control experiments, i.e., uncoated SS-MWs and NIP-MWs. It was observed that, for the bacteria counts between 0 and 105 CFU/mL, the CIP sensor did not have a significantly different response than the control experiments . However, the difference became more significant by further increasing the bacteria count to 106–109 CFU/mL, demonstrating the dominant effect of CIP coating as compared to NIP coatings or no coating at all. As shown in p-value < 0.01) than the sensor with uncoated SS-MWs . This can be because of the non-specific adsorption of bacteria to the NIP coatings [To further characterize the performance of the CIP-MW-based sensor, the effect of bacteria count on the normalized inter-wire resistance shift of the sensor was studied. coatings  that can0 curve established for the CIP-MW-based microfluidics sensor for bacteria counts from 0 to 109 CFU/mL. As can be seen, the dose–response curve of the sensor was non-linear; there were an insignificant response at bacteria concentrations below 104 CFU/mL and a saturation in the normalized resistance shift for bacteria counts higher than 107 CFU/mL. The former effect may have been due to the sensor insensitivity, and the latter effect was due to the high number of bacteria cells surpassing the CIP cavities [4–107 CFU/mL due to the significant statistical difference between the subsequent readout signals of the sensor in this range. A linear fit in this range with a goodness of R2 = 0.93 resulted in a sensitivity of 7.35 μS per CFU/mL, which is in the range of sensitivities achieved in similar works for the conductometric detection of E. coli bacteria using antibodies [5 CFU/mL and 7.3 × 105 CFU/mL, respectively.cavities . The dyntibodies  and aptatibodies . Based o5–106 cells/mL [The achieved detection range and limit with our established conductometric CIP-MWs-based sensor are comparable with other electrochemical methods for the detection of bacteria using CIPs . Howevercells/mL . NeverthListeria and Sarcina cells were used as the target microorganism, while the template for CIP preparation was E. coli.E. coli as the target microorganism results in a significant  drop in the normalized resistance of the sensor after washing the microchannel with electrolyte 2. However, in the case of using non-template microorganisms as the target, i.e., Listeria  and insignificant , respectively. Considering the size and shape of these two strains, where Listeria cells are rod-shaped with approximately 0.5 μm diameter and 0.5–2.0 μm length and Sarcina cells are spherical with 1.8–3 μm diameter [Listeria cells could be attributed to scarce capturing of these cells by E. coli cavities, which are similar in shape and size to the Listeria cells rather than the Sarcina bacteria. In conclusion, we concluded that our sensor is specific to the bacteria strain, even if cells with similar shapes and morphologies are used in the sensor. In the future, we aim to perform more complex and competitive assays with multiplex water samples.To investigate the specificity of the CIP-MWs in the developed sensor, Listeria B and Sar.A low-cost and miniaturized CIP-based microfluidic sensor for the conductometric detection of bacteria in water was designed and fabricated, and its sensing capacity toward target and non-target bacteria was assessed. The sensor comprised a pair of CIP-MWs installed perpendicularly to a microchannel. The inter-wire DC electrical resistance of the device was shown to significantly decrease after running bacteria suspensions for 30 min through the microchannel in comparison to the corresponding NIP-based sensor. Employing the DC approach eliminated complications associated with AC electrochemical methods such as the need for a reference electrode or selecting an appropriate equivalent circuit model for analyzing the frequency-dependent impedance in the electrochemical impedance spectroscopy (EIS) method, which could cause non-accurate results in electrode–electrolyte systems. Using bacteria suspensions with counts in the range of 0 to 10


BRCA 1/2. A total of 44 patients were enrolled, and the median follow-up duration was 22.9 months (interquartile range: 17.4–24.7). The primary outcome was 6-months progression-free survival (PFS), which was 88.6% , meeting the pre-specified primary endpoint. The secondary outcomes reported here include median PFS, 12-months PFS, and overall survival and safety. The median PFS was 22.4 months (20.4–∞), with a 12-months PFS rate of 84.0% (95% CI 69.3–92.0). The median overall survival was 28.6 months (27.3–∞). The combination demonstrated tolerable toxicity with manageable side effects. Other secondary outcomes include time-to-progression, time to subsequent treatment, time to second treatment and PFS2; however, this data is not reported, as treatment is still ongoing in a majority of patients. Exploratory analysis shows that patients who were homologous recombination deficiency-positive or had a programmed death-ligand 1 combined positive score ≥1 showed a favorable response . Thus, triplet maintenance shows durable efficacy with tolerable safety in patients with platinum-sensitive recurrence.In this multicenter, open-label, single-arm, Phase II study with Simon two-stage optimum design (NCT04361370), we investigate the efficacy and safety of triplet maintenance  in patients with platinum-sensitive recurrent ovarian cancer who are wild-type for  Even in patients who are initially sensitive, patients treated with platinum-based therapies often go on to relapse and have limited treatment options. Here, the authors report the efficacy and safety of a phase II trial investigating olaparib, pembrolizumab and bevacizumab as maintenance therapy in platinum-sensitive recurrent ovarian cancer. Once relapsed, patients inevitably follow the relentless disease trajectory hallmarked by increased resistance to therapy and shortened time to recurrence. The treatment for ovarian cancer is determined based on the treatment-free interval since the last platinum agent, and accordingly, patients are classified as having platinum-sensitive (relapse ≥6 months) or platinum-resistant (relapse <6 months) disease2. The standard of care for patients with platinum-sensitive recurrence is platinum-based chemotherapy3. However, repeated exposure to platinum agents causes toxicity and, ultimately, therapy resistance.Patients with ovarian cancer who have received primary surgery followed by platinum-based chemotherapy will most likely experience disease recurrenceBRCA mutation status6; this has led to PARP inhibitors being approved by the health regulatory agencies in the US7, Europe8, China9, and Korea10. However, across all studies, their greatest benefit was reported in patients with BRCA mutations, with limited activity observed in BRCA wild-type patients11. Another approved maintenance option for platinum-sensitive recurrence is bevacizumab, an antiangiogenic agent. However, the median PFS gain from adding bevacizumab was 3.4 months in GOG-21312 and 4.0 months in the OCEANS trial13. Outcomes from these historical trials suggest that the use of antiangiogenic agents as monotherapy may be insufficient for recurrent disease. Therefore, studies to identify optimal treatments for BRCA wild-type patients with platinum-sensitive recurrent ovarian cancer are required.In the platinum-sensitive recurrent cancer setting, maintenance with poly(ADP-ribose) polymerase (PARP) inhibitors was found to significantly improve progression-free survival (PFS) regardless of the BRCA wild-type patients with ovarian cancer, various PARP inhibitor-based combinations have been suggested. The first is olaparib plus an antiangiogenic agent. The combination of olaparib plus cediranib showed an improved outcome in BRCA-wild-type patients with platinum-sensitive recurrent ovarian cancer when compared to olaparib alone; this may have been because cediranib led to the induction of homologous recombination deficiency (HRD)14. Furthermore, in the frontline maintenance setting, patients receiving maintenance with olaparib plus bevacizumab showed a significant PFS benefit compared to bevacizumab alone in BRCA-wild-type, HRD-positive patients, thus expanding the potential pool of beneficiaries for olaparib15. Another potential PARP inhibitor-based combination is olaparib with an immune checkpoint inhibitor (ICI), such as the anti-PD-L1 or anti-PD-1 agents, and the combination of durvalumab and olaparib has shown promising activity with manageable toxicity in recurrent ovarian cancer17.To improve the outcomes for 18, suggest that combining PARP inhibitor with an ICI and antiangiogenic agent in the maintenance setting may enhance the efficacy of PARP inhibitor monotherapy in BRCA wild-type patients with ovarian cancer. Several ongoing phase III trials, namely DUO-O (NCT03737643), KEYLYNK-001 (NCT03740165), and FIRST (NCT03602859), are exploring the triplet combination as maintenance therapy in a frontline setting. In this trial, we evaluated the efficacy and safety of triplet maintenance therapy in BRCA wild-type patients with platinum-sensitive recurrent ovarian cancer.The aforementioned clinical studies, along with scientific research on the mechanismsBetween October 20, 2020, and March 22, 2022, 44 patients were enrolled in the study and treated accordingly Fig. ; their bThe study met the pre-specified primary endpoint, with a 6-month PFS rate of 88.6%  75.4–96.2). At the data cutoff point, 19 patients showed disease progression after a median of 13.7 months (IQR 8.6–20.8). Secondary endpoints were also investigated. Overall, the median PFS was 22.4 months (20.4–∞) Fig. . The 12-The treatment overview for each patient, including the first platinum-free interval and duration of triplet maintenance therapy, is shown in Fig. All patients experienced at least one adverse event (AE) of any grade. The summary statistics for AE are shown in Supplementary Table N = 8], anemia [N = 7], dyspepsia [N = 6], and nausea [N = 5]). With respect to each drug, dose interruptions were required in 38 patients (86.4%) for any of the three drugs, and in 32 patients (72.7%) for olaparib, 34 patients (77.3%) for pembrolizumab, and 33 patients (75.0%) for bevacizumab. Four patients permanently discontinued taking bevacizumab due to side effects  and continued the study with pembrolizumab and olaparib as per the study protocol.Twenty-seven (61.4%) of the 44 patients required a dose reduction for olaparib owing to an AE  of the 44 patients experienced grade 3 immune-related AEs, including alanine aminotransferase increase [N = 1], blood thyroid stimulating hormone increase [N = 1], cellulitis [N = 1], diabetes mellitus [N = 1], an abnormal liver function test [N = 1], myalgia [N = 1], and rash [N = 1], and shingles [N = 1]. No grade 4 immune-mediated AEs were observed.Immune-mediated AEs were reported in 36 (81.8%) of the 44 patients. The most frequent immune-related AEs that were causally associated with pembrolizumab were thyroiditis [Overall, there were no newly identified AEs or immune-related AEs, aside from the type and frequency of events that could be expected from each agent based on previous reports. All events were managed conservatively and appropriately. Aside from one patient with MDS, there was no case of discontinuation from the study owing to AEs or treatment-related deaths.P = 0.043); those with a PD-L1 CPS ≥1 showed improved PFS when compared to those with a PD-L1 CPS < 1 (P < 0.001). No significant difference was found regarding the response after second-line chemotherapy. A treatment overview plot stratified according to PD-L1 and HRD status is shown in Supplementary Fig. As exploratory outcomes, stratification was performed according to the pre-specified biomarkers  and a 12-months PFS of 84.0% (95% CI 69.3–92.0). The safety profile for the triplet combination was consistent with the known safety profiles expected for each agent individually.The OPEB-01 study investigated triplet maintenance with olaparib, pembrolizumab, and bevacizumab in BRCA wild-type platinum-sensitive recurrent ovarian cancer, with a median PFS of 15 months19. The most pronounced difference was that a triplet combination was used as a treatment in the MEDIOLA study and as maintenance in our study. Another difference was that the MEDIOLA study screened for patients based on germline BRCA status, whereas our study fully screened for both germline and somatic BRCA. In this study that exclusively included BRCA wild-type patients with platinum-sensitive recurrent ovarian cancer, the median PFS was 22.4 months. However, further maturation of the PFS data is necessary to elucidate the magnitude of benefit in maintenance versus treatment setting.The recently presented MEDIOLA study showed the promising efficacy of a triplet combination  as a treatment strategy for germline BRCA wild-type, platinum-sensitive recurrent ovarian cancer. In the OPINION trial, which investigated olaparib maintenance monotherapy in 279 patients without the germline BRCA mutation, the median PFS was 9.2 months (95% CI 7.6–10.9)11. There are two randomized trials involving PARP inhibitor monotherapy maintenance in BRCA wild-type patients: Study 1920 for olaparib and NOVA5 for niraparib maintenance in platinum-sensitive recurrent disease5. In the placebo groups of these two studies, the median PFS was consistently less than 6 months: 5.5 months for Study 19 and 3.9 months for NOVA. In comparison, in the PARP inhibitor maintenance subgroup, the median PFS was 7.4 months in Study 19 and 9.3 months in NOVA, translating into an absolute benefit of 1.9 months  in Study 19 and 5.4 months  in NOVA for PARP inhibitor compared to placebo. Furthermore, based on the PFS curves of these trials, the 12-months PFS rates were approximately 30% in the PARP inhibitor monotherapy group and 10% in the placebo group in these two trials. These findings are in contrast with the results from the OPEB-01 study, where the 12-month PFS rate was 84.0%. Overall, compared to monotherapy or doublet trials, the outcomes of our study suggest a potential synergy among the three different agents with an extension of the median PFS in a recurrent BRCA wild-type cohort beyond the benchmark of 19.1 months for patients with germline BRCA mutations in the SOLO-2 trial21.The efficacy of OPEB-01 can be compared to previous studies on currently available monotherapy options, namely PARP inhibitor and bevacizumab maintenance trials involving BRCA wild-type subgroup of the PAOLA-1 study, which was a frontline maintenance study with doublet regimen (olaparib and bevacizumab)15. DUO-O, a randomized, placebo-controlled phase III trial, showed a significant improvement in PFS with first-line chemotherapy with durvalumab and bevacizumab, followed by maintenance durvalumab, bevacizumab, and olaparib compared with control in patients with BRCA wild-type ovarian cancer22. The median PFS in DUO-O in the triplet maintenance arm was 24.2 months from the randomization. Direct comparisons between DUO-O and our study need to be interpreted with caution due to the differences in study design and the line of therapy. However, as shown in our study, the DUO-O study showed the efficacy of the triplet combination.Furthermore, our efficacy outcome surpassed the median PFS of 18.9 months in the somatic 21. Although the rate of immune-mediated AEs (81.8%) was higher in our study than the reported rate of 22.6% in the Keynote 100 study23, the events were mostly mild (grade 1 or 2). One of the most common immune-related AEs in our study was thyroiditis (20.5%), which was thyroid-related and thus similar to the most common AE in the Keynote 100 study, which was hypothyroidism (10.1%). Overall, the AEs and immune-related AEs were in line with those observed previously in the respective monotherapy studies, showing no evidence of drug–drug interactions among the three agents.The toxicity profile in our study was in line with that of previous studies. The most common AEs were hematologic toxicities, including anemia  and neutropenia . Both the toxicity rate and profile were similar to those in previous studies on olaparib monotherapy 15 or 65% in ATALANTE24. Similarly, our dose reduction rate of 61.4% was also higher than the 41% observed in PAOLA-1. There could be potential reasons. First, since all patients in our cohort were Asian, there could be ethnic differences. Second, we managed to achieve a low discontinuation rate through active dose reduction or interruption. In contrast, other studies frequently experienced discontinuation of the study drugs, such as 32.3% in MEDIOLA19 with a median follow-up of 31.9 months and 26% in DUO-O22 with a median follow-up of 23.3 months, whereas our study observed a discontinuation rate of 11.4%. Third, the triplet regimen could be associated with higher toxicity compared to mono or doublet regimen. However, the safety profile was generally consistent with that of the previous triplet regimen (DUO-O)22. The rate of AEs leading to dose modification was 76% in DUO-O (dose interruption rate was not reported), and our AE profiles were also similar.In terms of AE-related statistics, our study had high dose reduction and interruption rates, 61.4% and 86.4% (for any of the three study drugs), respectively. These rates were higher than those reported in previous studies on doublet regimen. For instance, our dose interruption rate of 86.4% surpassed 54% in PAOLA-1BRCA wild-type patients. A previous phase II study with olaparib and durvalumab (anti-PD-L1) in BRCA wild-type patients with platinum-sensitive recurrence showed that VEGFR and PIGF expression was significantly increased in biopsy samples while the patients were receiving the PARP inhibitor17. Such compensatory increases in VEGF may lead to therapy resistance via decreased T-cell function and trafficking and increased PD-1 expression in CD8 T-cells18. Thus, adding antiangiogenic inhibitors may help relieve the potential cause of therapy resistance. The consistent activity of triplet combination across three studies, MEDIOLA19, DUO-O22, and our study, further supports this hypothesis.In terms of activity, previous clinical studies have suggested that a triplet combination  may be more effective than a doublet combination (PARP inhibitor and ICI), especially in BRCA wild-type patients. Similar to the previous report from the PAOLA-1 study, our study observed longer PFS in patients with BRCA wild-type showing HRD tumors10. With respect to the PD-L1 status, our subgroup analysis suggested that patients with PD-L1 CPS ≥ 1 may benefit more from triplet maintenance than do those with PD-L1 CPS < 1, an observation that could be expected from the Keynote 100 study23. These are interesting aspects that could help form a hypothesis for large, phase III randomized trials.In addition to improving the efficacy, our data have suggested that triplet maintenance therapy may help expand the potential target population beyond BRCA status prior to enrollment. Pre-specified biomarkers, including HRD and PD-L1 status, were also assessed in most patients.Our study was limited by the fact that it was a single-arm, open-label study with a relatively small patient population and no comparator group. In terms of study design, we enrolled patients who had responded to second-line chemotherapy, making our cohort more favorable compared to previous studies where patients were enrolled regardless of their response to chemotherapy. Therefore, caution should be exercised when comparing our results with other maintenance trials, such as those involving bevacizumab, where the agent is administered concurrently with chemotherapy followed by maintenance, regardless of the response to chemotherapy. The 6-month PFS rate was chosen as the primary endpoint because this was a single-arm phase II study that evaluated signals for quick decision-making; based on previous randomized PARP inhibitor monotherapy trials, we expected that a majority of the patients would show recurrence within 6 months without maintenance therapy. However, it would be beneficial to have further survival maturation to determine whether the signals of durable responses translate into an overall survival benefit. Another limitation of our study is the small sample size, which was especially limiting for subgroup analysis of PFS concerning HRD or PD-L1 status. Additionally, we lacked an olaparib or bevacizumab monotherapy group as a comparator. Hence, a future randomized trial with triplet maintenance may be necessary. With these limitations in mind, the strength of our study is the homogenous patient population in a platinum-sensitive recurrent setting. All patients were screened for germline and somatic BRCA wild-type patients with platinum-sensitive recurrent ovarian cancer. Further research on biomarkers such as tumor microenvironment and RNA sequencing in pre- and post-treatment biopsies will be necessary to assess the specific mechanism of response and identify the patient subsets that would benefit most from triplet maintenance therapy. The long-term outcomes of triplet maintenance therapy will need to be further explored with survival maturation and additional randomized studies.In conclusion, findings from the OPEB-01 study show that the triplet maintenance therapy with olaparib, pembrolizumab, and bevacizumab leads to promising outcomes and is tolerable in BRCA Non-mutated Patients With Platinum-sensitive Recurrent Ovarian Cancer (OPEB-01)”  on April 2020.The trial was conducted in accordance with the Declaration of Helsinki and the Guidelines for Good Clinical Practice. The trial was approved by the institutional review board of each institution . Written informed consent was obtained from all participants before study enrollment. Patients did not receive any compensation for their participation. The trial was registered under the name “Olaparib Maintenance With Pembrolizumab & Bevacizumab in 25. The first patient was enrolled on October 22, 2020, and the last patient was enrolled on March 22, 2022. Eligible patients were women ≥ 20 years of age, with an Eastern Cooperative Oncology Group performance status of 0 or 1, histologically confirmed epithelial ovarian cancer, and lacking germline and/or tumor BRCA mutations. Gender was not considered in the study design since this trial was on women’s cancer. With respect to histology, patients with high-grade predominantly serous, endometrioid, carcinosarcoma, mixed Mullerian with high-grade serous components, clear cell, or low-grade serous ovarian cancer, primary peritoneal cancer, or fallopian tubal cancer were considered. A cap of eight patients was applied for clear cell carcinoma; mucinous carcinoma could be enrolled. Patients had received two previous courses of platinum-containing therapy and showed platinum-sensitive disease  following their penultimate platinum course, along with a complete response (CR) or PR to their most recent platinum course; they were enrolled in the study within eight weeks of completing their final platinum regimen, regardless of prior PARP inhibitor or bevacizumab use but had to be immunotherapy naïve. The full eligibility criteria are presented in the study protocol (Supplementary Note).OPEB-01/Asia-Pacific Gynecologic Oncology Trials Group (APGOT)-OV4 is an investigator-initiated, multicenter, single-arm, open-label, phase 2 study that was conducted in five medical centers across Korea and Singapore  and bevacizumab , followed by a combination of 300 mg olaparib twice daily (up to two years and longer in case of PR at two years), 200 mg pembrolizumab every 3 weeks (cycles 2 through 35), and 15 mg/kg bevacizumab every 3 weeks intravenously until progression or intolerable toxicity. Unlike olaparib and bevacizumab, which were started in cycle 1, pembrolizumab was initiated in cycle 2, based on the preclinical rationale that PARP inhibitors induce immune cell infiltration and PD-L1 upregulation, leading to enhanced antitumor immunity that can be further enhanced through the combination of an immune checkpoint inhibitor. Patients were allowed to withdraw from the study at any time.26.Dose modifications to manage toxicities were allowed. Olaparib toxicities were managed with supportive care, dose interruptions, or dose reductions . If a patient could not tolerate olaparib at 200 mg twice daily, the patient had to be discontinued. Dose re-escalation was also not permitted, but dose interruptions of less than 4 weeks were permitted. Hematotoxicity was monitored and managed as specified in the protocol (Supplementary Note). With respect to AE reporting, we have adhered to the exact terms used by clinicians. Pembrolizumab and bevacizumab toxicities could be managed with supportive care or dose interruptions; dose reductions were not permitted. Patients were discontinued if pembrolizumab was interrupted for 12 weeks or longer due to AEs or toxicity or for ≥3 weeks due to administrative causes. Bevacizumab was considered a background therapy; its administration was based on the clinicians’ discretion, and patients were allowed to continue with olaparib and pembrolizumab if bevacizumab was interrupted or discontinued. Prophylaxis for nausea and vomiting was not mandatory but was allowed. Tumor assessment was performed using computed tomography or magnetic resonance imaging of the chest, abdomen, and pelvis every three cycles for the first 2 years, every four cycles from the second to the third year, and every six cycles from the third year onward. Assessments were performed up to 7 days before or after the designated time point by the investigator using the Response Evaluation Criteria in Solid Tumours version 1.123, and HRD status for olaparib was determined based on the PAOLA-1 study15. Immunohistochemistry (IHC) was performed using a Ventana Benchmark XT automated stainer  with antibodies against PD-L1 . PD-L1 expression in the tumor cell membrane and the membrane and/or cytoplasm of tumor-associated mononuclear inflammatory cells was scored. The CPS was defined as the total number of tumors and immune cells stained with PD-L1 divided by the number of all viable tumor cells and then multiplied by 100. Genomic scarring was estimated by determining copy number alterations in the whole exome sequencing data using Sequenza-utils (v.3.0.0)27, based on the loss of heterozygosity, large-scale transitions, and the number of telomeric allelic imbalances, and these were estimated using the scarHRD (R package v.0.1.1)28. The sum of these values served as the genomic scar score and was used as the input seqz file31. Based on the genomic scar score and a cutoff of 42, HRD status was determined.For biomarker analysis, archival tumor tissues were collected from all patients. These biomarkers were pre-determined based on previous reports on monotherapy. For instance, PD-L1 was considered a biomarker for pembrolizumab based on the Keynote-100 studyThe primary endpoint was the 6-month PFS rate. PFS was defined as the time from the start of treatment to the first documented sign of disease progression or death from any cause. The reported secondary endpoints included PFS, OS, and safety. Other secondary endpoints, such as time time-to-progression, time to subsequent treatment, time to second treatment, and PFS2 were not reported because a majority of patients were still ongoing at data cutoff. OS was defined as the time from the first treatment to death from any cause. The cutoff date was May 25, 2023. Investigation of biomarkers of response was a pre-specified exploratory outcome.BRCA mutations. Recognizing the conceivable differences between GOG-213 and our trial, which focuses on the maintenance therapy, we used the best approximation from GOG-213 by considering the chemotherapy time window because of the lack of data on studies with bevacizumab maintenance in patients responding to chemotherapy. Thus, based on the current standard of care and the best approximation from GOG-213, the rate of patients with a disease-free state at 6 months was expected to be 50% with bevacizumab maintenance. Moreover, the HR of adding maintenance therapy with a triplet combination  was assumed to be 0.5, equivalent to a PFS rate of 70.7%. The null hypothesis for this study would be a 6-month PFS rate of 50%, and the alternative hypothesis of interest would be a 6-month PFS rate of 70%. Using Simon’s two-stage optimal design at a one-sided 5% level of significance and 80% power, 39 patients were included in this study. In the first stage, 22 patients would be enrolled; if 10 or more PDs were observed, the trial would be terminated. Else, the trial would continue to the second stage. The null hypothesis would be rejected if the total number of PDs was less than 15. Considering loss to follow-up, the 44 patients would be studied.The study was conducted using Simon’s two-stage optimal design with assumptions concerning the estimated PFS rate in ovarian cancer. As the benchmark for the null hypothesis, we chose the GOG-213 study, which investigated chemotherapy plus bevacizumab followed by bevacizumab maintenance regardless of The proportion of patients achieving responses and 95% CIs was assessed using the Clopper–Pearson exact method. Survival analyses were pre-specified as secondary endpoints. The PFS and associated 95% CIs were calculated using the Kaplan–Meier method. A log-rank test was used to compare the PFS between the patient subsets. Statistical analyses were performed using SAS .Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data file


A chemotherapy of rituximab, fludarabine and cyclophosphamide (R-FC) has been accepted as a promising frontline chemotherapy in selected patients with chronic lymphocytic leukemia (CLL). Although R-FC regimen is a relatively dose-dense regimen and neutropenia incidence is more than 50%, primary prophylactic pegfilgrastim was not fully recommended in the clinical field. Therefore, the study evaluated the prophylactic effectiveness of pegfilgrastim to reduce the incidence of febrile neutropenia associated with R-FC of patients with CLL.A single-arm, multicenter, prospective phase II study was designed to assess the efficacy of prophylactic pegfilgrastim. Thirty-four CLL patients were enrolled and analyzed for neutropenia and other related factors, and comparative analysis was performed with historical cohort.Compared with our historical cohort, incidence of grade 3-4 neutropenia and febrile neutropenia was remarkably reduced during any cycle of chemotherapy . Also, cumulative incidence of disrupted chemotherapy was noticeably reduced in study cohort on any cycles of R-FC regimen . In addition, treatment-related mortality was 5.9%, which significantly reduced compared to 9.6% of our historical cohort .Primary prophylactic pegfilgrastim is effective in the prevention of neutropenia/febrile neutropenia, and infection-related mortality during R-FC regimen in patients with CLL. Chronic lymphocytic leukemia (CLL) is the most common lymphoid malignant disease in adults in western societies, with a prevalence of approximately 4.7 cases per 100 000 population per year, corresponding to more than 20,100 new cases per year .P < 0.0001) and a favorable overall survival (OS) rate  , spleen tyrosine kinase (SYK), and phosphoinositide 3-kinase (PI3K) inhibitors that disrupt the B cell receptor (BCR) signaling pathway, and venetoclax, an antagonist of the antiapoptotic protein BCL-2 , 5. Alth3 and at least one temperature measurement of ≥ 38°C  of 90%–95% and rate of complete remission (CR) of 40%–70% as 3-year survival outcomes. However, an increased incidence of hematological complications during R-FC therapy was reported along with these favorable survival outcomes. Grade 3 or 4 neutropenia occurred at a rate of 34%–85%, the incidence of major infections was 2.6%–16% per chemoimmunotherapy cycle, and the minor infection rate was 10%–38% per course  . HoweverA total of 51 patients with newly diagnosed untreated CLL between April 2016 and October 2018 were screened for inclusion in this study; 34 patients were enrolled Figure 1Patients were excluded if they had a known grade > 3 hypersensitivity to pegfilgrastim, active uncontrolled infection, HIV-positive patients, active uncontrolled hemorrhagic status, active uncontrolled medical conditions , severe congestive heart failure, acute myocardial infarction within 6 months before the screening period, Patients with severe or uncontrolled medical conditions , Other clinical trials , women who were pregnant or nursing, and patients who are judged not to be suitable for the progress of the study by the judgment of other clinical trial directors Figure 1After chemotherapy cycle 2, although there were no hematological or nonhematological adverse events, three patients discontinued treatment because they did not wish to receive more chemotherapy, and one patient stopped chemotherapy due to a CLL-unrelated cerebral hemorrhage after cycle 3. In addition, one patient abandoned chemotherapy after each of cycles 5 of R-FC due to poor general performance and weakness without hematological toxicity. These five patients were also used in the analysis of the pegfilgrastim effect in this study.Historical cohort was defined as patients who were diagnosed with CLL by examining the medical records of each institution from January 2013 and treated with R-FC regimen for more than one cycle, and compared with this study cohort.®; Kyowa Kirin Korea Co., Ltd., Seoul, Korea) administration during the full course of R-FC regimen in patients with CLL. R-FC chemotherapy was administered as follows: on day 1 of cycle 1, 375 mg/m2 of rituximab was administered and 25 mg/m2/day of fludarabine with 250 mg/m2/day of cyclophosphamide on days 2, 3, and 4. In cycles 2 to 6, the rituximab dose was increased to 500 mg/m2 on day 1. Fludarabine and cyclophosphamide were administered on days 1, 2, and 3. Courses were repeated every 4 weeks, depending on the recovery of blood counts, and were delayed until the platelet count exceeded 80 × 106/L and the ANC exceeded 1.0 × 109/L. In addition, the doses of fludarabine and cyclophosphamide were reduced if blood counts had not recovered to the levels described above 5 weeks after the last course of therapy or if major infections occurred. Pegfilgrastim was injected subcutaneously at a dose of 6 mg in a single administration ~24 h after completion of each cycle of R-FC. Blood tests and basic physical examinations were performed every week on an outpatient basis.This was a multicenter, open-label, non-randomized, non-comparative, phase II study which designed to determine the efficacy and safety with regard to neutropenia/febrile neutropenia of once-per-cycle pegfilgrastim .The study protocol was approved by the institutional review boards and local ethics committees of all participating centers. All eligible patients gave written informed consent before any study-related procedures. This study was registered with the Clinical Research Information Service .Since the main purpose of this study was to determine whether the use of pegfilgrastim during administration of R-FC chemotherapy could reduce the incidence of grade 3-4 neutropenia: The primary efficacy endpoints were the incidence and duration of grade 3 or 4 neutropenia, defined as an ANC < 1.0 × 10The sample size of this clinical study was based on hypothesis testing, type I error, and power considerations. The incidence rate of febrile neutropenia in the preemptive treatment group was entered as 1% and the incidence rate in the standard treatment group as 10%, 15%, and 20%, respectively, and was calculated. As a result, at least 31 patients were calculated, and a plan was made to recruit a total of 35 or more to consider a dropout rate of 10%. Incidence of neutropenic fever was calculated using the efficiency–one proportion vs. a given proportion: conditional method.t test. The least squares mean difference and 95% confidence interval (CI) between two groups were determined. Secondary endpoints were derived from two-sided stratified and non-stratified log-rank tests for time-to-event outcomes. In these analyses, P < 0.05 was taken to indicate statistical significance. For safety analyses, the number and ratio of patients experiencing adverse events were summarized, and comparisons between groups were performed using the Fisher’s exact test and χ2 test for categorical variables and the Mann-Whitney test for continuous variables. OS was calculated from the date of the start of R-FC cycle 1 until the date of death from any cause or the last follow-up. disease-free survival (DFS) was defined as the time from the date of the start of R-FC cycle 1 until the date of disease relapse/progression, death from any cause, or the last follow-up. The OS and DFS rates were calculated using the Kaplan-Meier survival method with log-rank analysis. The cumulative incidence of relapse (CIR) was calculated with relapse or death from other causes defined as adverse events using the Gray test for univariable analysis and the Fine-Gray method for proportional hazard regressions. All statistical analyses were performed using R version 3.2.0  with the EZR graphical user interface by Y. Kanda   .n = 16, 47.1%) were in an advanced Rai stage (stage III or IV) and the majority of patients  were in a moderate- to high-risk Binet stage (stages B and C). Although the number was small , the cohort included patients with poor prognostic factors, such as del(17p), which are known to have negative impacts on survival outcomes.The clinical baseline characteristics of all patients in the study cohort are summarized in For an indirect comparison, the study cohort enrolled in this research was compared with the historical cohort that had been treated in the past by these institutions. There was no difference in the median age between the two groups as about 60 years old (p=0.866), but the male proportion in the study cohort was 63%, which was smaller than 70% of the historical cohort (p=0.034). In addition, there was no difference between the two groups in laboratory test including of white blood cells, hemoglobin, platelets and LDH etc. as well as Rai/Binet stage at initial diagnosis. All patients in the study cohort were previously administrated prophylactic pegylated filgrastim, and patients with historical cohort received non-pegylated filgrastim for the therapeutic purpose of neutropenia Table 1.n = 18) showed a median of 4 days of neutropenia. Febrile neutropenia was most common in cycle 1 of chemotherapy, and four cases (2.2%) showed a median of 4 days of febrile neutropenia out of the total of 186 cycles of chemotherapy , five patients (17.9%) had a disrupted chemotherapy schedule due to severe neutropenia or febrile neutropenia . Disrupted chemotherapy consisted of time-disrupted and dose-disrupted chemotherapy; the details of each cycle are presented in 9/L, filgrastim was administered for therapeutic purposes after R-FC infusion in the historical cohort.Although this was a single-arm study to determine the efficacy of pegfilgrastim for CLL with the R-FC regimen, indirect comparisons were performed with historical data from CLL patients who had been treated previously at our hospital to determine differences in efficacy with a nonprophylaxis group. As the use of prophylactic pegfilgrastim after R-FC was not approved according to national reimbursement standards, it could not be used previously; therefore, in cases in which the ANC decreased below 1.0 × 10P < 0.001) (P = 0.004) (P = 0.007) .This was a phase II prospective study conducted without a placebo control group from the beginning. However, it was necessary to compare survival outcomes using the historical cohort from our center to determine the efficacy of pegfilgrastim prophylaxis for neutropenia in a cohort of the same ethnicity treated with a similar therapeutic strategy. The median follow-up period from the start of cycle 1 of R-FC was 2.6 years (range: 3 months to 3.7 years) in the study cohort and 5.9 years (range: 2 months to 14.2 years) in the historical cohort. There were generally fewer deaths and adverse events among patients receiving R-FC. When examining the rate of early mortality, defined as all-cause mortality during the chemotherapy period, it was 5.9% (2/34) in the study cohort, which was significantly reduced compared to 9.6% (8/83) of the historical cohort   Figure 3In this prospective study, we investigated the role of primary prophylaxis with pegfilgrastim as long-acting G-CSF in a cohort of 34 patients with CLL receiving R-FC. The incidence of grade 3 or 4 neutropenia was 9.7% (18/186 cycles) and that of febrile neutropenia was 2.2% (4/186 cycles). In addition, the cumulative incidence of disrupted chemotherapy was 8.8% at cycle 2 and reached 17.9% at the last cycle (cycle 6) of chemotherapy. These results showed that the incidences of grade 3 or 4 neutropenia and febrile neutropenia were significantly decreased in comparison with our historical cohort treated without pegfilgrastim prophylaxis. In addition, pegfilgrastim prophylaxis reduced the rate of a delayed chemotherapy schedule and dose reduction due to neutropenia. Although it has already been reported that the use of pegfilgrastim for primary prophylaxis rather than filgrastim reduced rates of febrile neutropenia, febrile neutropenia-related hospitalization, and intravenous antibiotic use in patients with breast cancer  , and thuOur study focused on R-FC chemotherapy for CLL patients. In general, the guidelines for the use of pegfilgrastim re commend that combination chemoimmunotherapy with a febrile neutropenia incidence of 20% or more is classified as high-risk . The R-FP = 0.002), and Vogel et al. (P = 0.0.38). These findings indicate the impact of primary pegfilgrastim prophylaxis on infection-related and all-cause early mortality as well as a reduction in disrupted chemotherapy.Although a comparison of survival outcomes is not entirely suitable for the purpose of this study, survival analyses were conducted to determine whether there were any changes in survival outcomes as a result of febrile neutropenia reduction and a decreased rate of disrupted chemotherapy with pegfilgrastim prophylaxis. The results indicate no differences in long-term OS, PFS, or CIR in comparison to the historical cohort, but we found a significant decrease in early mortality rate, including death due to a neutropenia-associated infection. Similar to our results, a meta-analysis by Kuderer et al.  reportedl et al.  demonstrOur study showed favorable results of pegfilgrastim prophylaxis in CLL patients receiving R-FC chemotherapy. However, this analysis had several limitations. First, the number of patients enrolled in the study was too small to draw definitive conclusions. In addition, the number of adverse events was small, and the study was not designed and did not have sufficient power to assess OS. Second, the efficacy and survival outcomes of prophylactic pegfilgrastim were examined by comparison of the study cohort with a historical cohort. However, this was not a direct comparison but an indirect incomplete comparison, and therefore care is required in interpreting the results. Third, the follow-up periods were not equivalent among the cohorts .In conclusion, this prospective study suggests that primary pegfilgrastim prophylaxis can reduce the rate of grade 3 or 4 neutropenia/febrile neutropenia in patients with CLL receiving R-FC chemotherapy in contrast to previous studies and our historical cohort in which chemotherapy dose adjustment and infusion schedule delays occurred frequently during R-FC therapy without G-CSF prophylaxis. Moreover, the reduction in the early mortality rate itself suggests that the use of pegfilgrastim prophylaxis is important during R-FC chemotherapy in patients with CLL. Although R-FC is not currently classified as high-risk for febrile neutropenia, considering the characteristics of CLL as an immunosuppressive disease as well as a dose-dense chemoimmunotherapeutic regimen R-FC, prophylactic pegfilgrastim support is an essential adjuvant for R-FC therapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol was approved by the institutional review boards and local ethics committees of all participating centers. The patients/participants provided their written informed consent to participate in this study.This manuscript has been seen and approved by all authors, whose individual contributions are as follows: YJ and professor S-GC were contributing to conception and design, acquiring data, analyzing and interpreting data, and drafting the manuscript. YJ and professor S-GC were critically contributing to or revising the manuscript. D-HY, S-JO, J-HP, J-AK, S-YK, C-WC, W-SL, I-HK, Y-CM, GM, and K-SE were enhancing its intellectual content. And S-GC was approving the final content of this manuscript.


Both weight regain and dumping syndrome (DS) after Roux-en-Y gastric bypass (RYGB) have been related to the dilation of gastro-jejunal anastomosis. The aim of this study is to assess the safety and long-term efficacy of endoscopic transoral outlet reduction (TORe) for DS and/or weight regain after RYBG.A retrospective analysis was performed on a prospective database. Sigstad’s score, early and late Arts Dumping Score (ADS) questionnaires, absolute weight loss (AWL), percentage of total body weight loss (%TBWL), and percentage of excess weight loss (%EWL) were assessed at baseline and at 6, 12, and 24 months after TORe.2. Out of 87 patients, 58 were classified as “dumpers” due to Sigstad’s score ≥ 7. The resolution rate of DS (Sigstad’s score < 7) was 68.9%, 66.7%, and 57.2% at 6, 12, and 24 months after TORe, respectively. A significant decrease in Sigstad’s score as well as in early and late ADS questionnaires was observed (p < 0.001). The median Sigstad’s score dropped from 15 (11–8.5) pre-operatively to 2 (0–12) at 24 months. The %TBWL was 10.5%, 9.9%, and 8.1% at 6, 12, and 24 months, respectively. Further, “dumpers” with resolution of DS showed better weight loss results compared with those with persistent DS (p < 0.001). The only adverse event observed was a perigastric fluid collection successfully managed conservatively.Eighty-seven patients  underwent TORe. The median baseline BMI was 36.2 kg/mTORe is a minimally invasive treatment for DS and/or weight regain after RYGB, with evidence of long-term efficacy.The online version contains supplementary material available at 10.1007/s11695-023-06466-w. Roux-en-Y gastric bypass (RYGB) is one of the most frequently performed bariatric surgeries and has proved excellent long-term outcomes in terms of weight loss and comorbidity improvement –3. HowevDumping syndrome (DS) consists of a cluster of symptoms induced by the rapid transit of undigested food into the small bowel. The dilation of the gastro-jejunal anastomosis has been related to DS, along with weight regain after RYGB , 11. AccThe aim of this study is to assess the safety and long-term efficacy of endoscopic transoral outlet reduction (TORe) for DS and weight regain after RYBG in a single tertiary center.A retrospective analysis was performed on a prospective database collecting data on patients who underwent TORe between January 2015 and June 2021 at the Digestive Endoscopy Unit of Fondazione Policlinico Universitario A. Gemelli IRCCS in Rome. The institutional ethical committee approved this clinical investigation . Informed consent was obtained from all individual participants included in the study. The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.Weight regain ≥ 50% of the weight loss after RYGBDS refractory to medical therapyEndoscopic evidence of gastro-jejunal anastomosisPre-operative assessment by local bariatric multidisciplinary team with indication to endoscopic revision.The inclusion criteria were:All patients who did not fulfil inclusion criteria and those with other types of bypass  were excluded from the analysis.2.Dumping syndrome’s presence and severity were assessed by Sigstad’s score Table  19]. Th. Th19]. All TORe procedures were performed under general anesthesia using the full-thickness suturing system Apollo OverStitch , and a double-channel therapeutic endoscope . Before suturing, the rim of the anastomosis was cauterized by argon plasma coagulation (APC) at a flow of 1 L/min and 40 W to mark the margins of the anastomosis and to enhance the suture tightening during the scarring process Fig. , Video 1p-value < 0.05 was considered statistically significant.Statistical analyses were conducted using the STATA version 16 software (STATA Corp). All variables included in the study were summarized using descriptive statistical techniques. Qualitative data were reported as absolute and percentage frequencies. As regards the quantitative variables, the distribution of the data was first verified by the Shapiro-Wilk normality test. Continuous variables were reported as means and standard deviations (SD) or as medians and interquartile ranges (IQR) in case of deviations from normality. The differences between baseline (TORe) and 6, 12, and 24 months were evaluated, as regards the quantitative variables, both overall, through the non-parametric Friedman test, and two by two  through the non-parametric Wilcoxon test for paired samples. Comparisons between qualitative variables, pre-post according to the above scheme, were performed by McNemar’s test. Comparison in weight loss parameters in patients with resolution of DS and patients with refractory DS after treatment was performed by the non-parametric Wilcoxon test. A Eighty-seven patients underwent TORe between January 2015 and June 2021 at our center. Baseline characteristics of the patients are summarized in Table All 87 patients (100%) completed the 6-month follow-up. Seventy-six patients (87.4%) completed the 12-month follow-up, 3 (3.4%) were lost to follow-up, and 7 (8%) were still waiting to complete the 12-month follow-up. Fifty-six patients (64.4%) completed the follow-up at 24 months, 7 (8%) were lost to follow-up, and 22 (25.3%) had yet to reach the 24-month follow-up. After a multidisciplinary assessment, TORe was repeated in one previously “non-dumper” patient due to the new onset of DS within 12 months and in two other “non-dumper” patients (2.3%) because of weight regain within 2 years. In total, 3 patients required a re-TORe and were excluded from the statistical analysis. To provide a scientifically robust evaluation of the variation over time of dumping syndrome’s parameters and weight loss outcomes after TORe, we focused statistical analyses on those patients who completed the follow-up at 24 months.One serious adverse event was observed: a perigastric collection, in a patient with a clinical history of post-traumatic splenectomy, occurred in the first 48 h. The patient complained persistent post-procedural abdominal pain and fever and was successfully treated with piperacillin-tazobactam (grade II sec. Clavien-Dindo classification). No procedure-related deaths and no other adverse events were observed.Fifty-eight out of 87 patients (66.7%) were classified as “dumpers” at baseline according to Sigstad’s score ≥ 7. Of these, 51 reached follow-up at 12 months and 35 reached follow-up at 24 months. Forty of 58 patients (68.9%), 34 of 51 patients (66.7%), and 20 of 35 patients (57.2%) showed resolution of symptoms (Sigstad’s score) < 7 at 6, 12, and 24 months, respectively. We observed a statistically significant decrease in all symptom-based scores for DS  may experience a progressive weight regain over the long term , 21, 22.The first step in the management of DS is dietary adjustments, with a reduced quantity of food at each meal and avoidance of simple sugars . As nextp = 0.001) [p-value < 0.001) as well as in both early and late ADS questionnaires . Eventually, we had no case of recurrence of DS in 2 years.The endoscopic transoral outlet reduction (TORe) is proposed as a minimally invasive treatment for patients with DS refractory to medical therapy and/or weight regain after RYGB , 18. The= 0.001) . Similar= 0.001) . Our stuRegarding weight loss outcomes after endoscopic revision of the anastomosis, a recent systematic review reported a mean weight loss of 6.27 kg, sustained up to 24 months, but the %EWL was 19.3% at 6 months and 10.3% at 24 months . Furtherp-value < 0.001) analyzing weight loss outcomes in “dumper” patients with resolution of DS compared to those with persistent symptoms after TORe. In more details, “dumper” patients that resolved DS showed overall better weight loss results compared to those with persistent DS. Probably, the resulting delayed emptying and the smaller amount of food to the small bowel also caused a change in their eating habits. We observed that patients with persistent DS, contrary to what was expected, often tend to eat sugars to mitigate the hypoglycemic symptoms.Further, in our analysis, the two-by-two comparisons  of both symptom-based scores for DS and weight loss outcomes showed no statistical difference between 6–12 and 12–24 months; this evidence suggests that the results obtained in the first months are maintained over 2 years. We did not observe any statistically significant difference in terms of weight loss comparing “dumper” and “non-dumper” patients. Interestingly, we found a statistically significant difference (One serious adverse event (1.1%) was observed, a perigastric collection that was effectively treated with antibiotic therapy. No procedure-related deaths occurred. TORe is a low-risk procedure, as highlighted in previous studies, where serious adverse events reported are < 1% , 26, 27.This study has some limitations that should be pointed out, including the retrospective design, the absence of a control group, and a possible selection bias resulting from the single-center counselling. Eventually, data at 2 years were available for only 64.4% of the treated patients. Nevertheless, as strengths, this is the first study evaluating not only the Sigstad’s score but also the Arts Dumping Score as tools to monitor the efficacy of TORe for DS. In more details, the impact of the procedure was analyzed by differentiating between early and late symptoms. Furthermore, this study has a longer follow-up compared with other studies in the literature.According to our experience, TORe is an effective minimally invasive treatment for both early and late DS along with weight regain after RYGB, with evidence of long-term maintenance of the results achieved in the first months. Given the good outcomes and the excellent safety profile, TORe may play a key role in the multidisciplinary approach to weight regain and/or DS after RYGB when conservative therapy fails and before surgical revision Fig. .Fig. 5FlESM 1:(MP4 108300 kb)


Background: One of the most prevalent malignancies in India is oral squamous cell carcinoma (OSCC), which is found in more than 90% of cancer cases and has a reduced survival rate of 30%. Matrix metalloproteinases (MMPs) are zinc-containing and calcium-dependent endopeptidases that regulate angiogenesis, migration, and proliferation. MMP-9 in OSCC increases tumor progression through angiogenesis, degrades the basement membrane, and facilitates metastasis by changes in tissue shape. Its overexpression in OSCC has also been shown to have prognostic significance.Aim: This study aims to evaluate the serum levels of MMP-9 in OSCC patients and healthy controls and to correlate with its clinicopathological staging.Materials and methods: This study included 40 individuals; 20 patients with OSCC and 20 healthy controls. MMP-9 was determined in serum samples utilizing enzyme-linked immunosorbent assays.Results: Descriptive statistics showed that 90% of the patients included in the OSCC groups were above 40 years, and 85% were males. There was a significant increase in the serum level of MMP-9 in OSCC patients compared to healthy controls with a mean difference of +28% (393.21 pg/ml) and a significant p-value of 0.001. (1365.80 ±236.414 pg/ml vs 973.67 ± 83.416 pg/ml). There was a significant increase in the serum levels of MMP-9 among the tumor stages and nodal involvement with a significant p-value of 0.002 and 0.001. No significant association was found between the age and gender groups in OSCC patients and serum levels of MMP-9.Conclusion: MMP-9 was significantly increased in OSCC when compared to healthy controls. Hence, MMP-9 can be used as a prognostic indicator in assessing tumor staging and nodal involvement. Matrix metalloproteinase (MMP) is a zinc-dependent endopeptidase that involves the degradation of collagen in the extracellular matrix. They play an important role in tissue repair and extracellular matrix (ECM) remodeling, which in turn promotes cancer invasion ,2. It isRecent literature provides well-established evidence regarding the overexpression of MMP-9 in cancer ,6. AdditAmong the cancers reported, oral cancer is the 6th most common type. Oral squamous cell carcinomas (OSCC) are the most common type of oral cancer, accounting for more than 90% of all cases ,11. InciMolecular and biochemical alterations might provide essential information for predicting tumor behavior, recurrence, and metastatic potential even when the surrounding healthy tissues of oral cancers may appear clinically normal (concept of field cancerization) ,6. ECM rRecently, the emphasis has been on finding molecular markers in human fluids including serum, saliva, and urine to diagnose the condition, determine its prognosis, and predict its progression . Since bAlthough various studies have been done to assess the immunohistochemical and gene expression of MMP9, few studies have reported the serum levels of MMP9 in OSCC in the Iranian population. Studies evaluating the serum levels of MMP-9 in OSCC patients in the Indian population are sparse .This study aims to evaluate the serum level of MMP9 in OSCC patients when compared to the healthy controls and to correlate with the clinicopathological findings. Study designThis was a cross-sectional study conducted in the Department of Oral Pathology after getting ethical clearance from Saveetha Dental College Institutional Human Ethics Committee . Following an explanation of the study and information about the sampling procedures, each subject signed a written informed consent form.Inclusion criteriaPatients were diagnosed with OSCC based on oral clinical examination and confirmed by histopathological examination. The controls were generally healthy people without any history of tobacco smoking or drinking alcohol and without any underlying systemic diseases, any cancer, or inflammatory oral lesions.Exclusion criteriaPatients with autoimmune disorders, hepatitis, inflammatory oral lesions, periodontal disease, systemic disease, prior malignancy or human immunodeficiency virus infection, as well as pregnant or breastfeeding women, were excluded from the study.Sample collectionThe sample size of 40 for our study was calculated utilising Gpower and epidemiological data of our region. Hence, 20 patients with OSCC were in the study group and 20 healthy individuals were included as controls. The controls were age-matched and gender-matched to the study group. After obtaining written consent, serum samples were collected from patients in the OSCC group and the control group Figure .The demographic details for all study participants were collected. The clinicopathological characteristics of tumors in patients with OSCC were recorded. Age, gender, tumor site, tumor stage as determined by the American Joint Committee on Cancer (AJCC) TNM classification, and histological tumor grade were collected Figure .ProcedureFour milliliters (ml) of fasting venous blood was drawn under stringent aseptic conditions from the cubital vein of study participants after they signed an informed consent form. Blood was drawn and placed in sterile tubes and vacutainers. It was allowed to be centrifuged for 10 minutes at 3000 rpm to separate the serum after two hours at room temperature. Until the levels of MMP-9 were measured, the serum samples were stored at 20°C.The serum MMP-9 concentrations were measured using RayBioTech Human MMP-9 enzyme-linked immunosorbent assay (ELISA) kit  Figure .The manufacturer's protocol was followed for analysis.Every assay was carried out twice. The kit used a sandwich ELISA method, in which the sample's MMP-9 binds to a polyclonal antibody that is specific to MMP-9 and is coated on a microplate. MMP-9 that has been attached to the plate is detected by a secondary, biotinylated MMP-9-specific antibody. At 450 nm, the intensity was measured. The final readings were calculated in pg/ml and a set of standards was loaded alongside. A trained and calibrated professional carried out the procedure.Statistical analysisThe serum levels of MMP-9 of our study were assessed using SPSS version 19  with a chi-square test and one-way ANOVA The correlation between MMP-9 levels and clinicopathological findings in patients with OSCC was assessed using Pearson's correlation. P value < 0.05 was considered to be statistically significant.This study included a total of 40 samples of which there were 20 patients with OSCC and 20 healthy controls. About 90% of the patients included in the OSCC groups were above 40 years, and nearly 85% were male. The control group included 50% of male patients and 50% of female patients. Among the SCC cases, 40% were in the buccal mucosa region, 30% in the gingivobuccal sulcus region, and 30% in the tongue Table .Considering the TNM classification, 60% of cases were in the T2 grade, and 40% of cases were in the T3 grade. Lymph node involvement was observed in 70% of the included cases with 35% in N1 and 35% in N2. There was no metastasis. The OSCC group has a higher level of serum MMP-9 when compared to healthy controls, with a mean difference of +28%(393.21) and a significant p-value of 0.001 (1365.80 ±236.414 VS 973.67 ± 83.416) . There was no significant difference in the serum level of MMP-9 in male and female patients. .Serum levels of MMP-9 increased statistically in T2 when compared to T1 (1618.93±172.86 vs 1328.32± 433.33) with a p-value of 0.002. There was a gradual increase in the serum levels of MMP-9 from N0 to N2  with a significant p-value of 0.001. In India, oral cancer is a major public health issue. Globocan reported that 13,24,413 new cases and 8,51,678 deaths were reported in India in 2020 . OSCC caThe metastatic potential of OSCC depends on its ability to degrade ECM, penetrate the basement membrane, initiate tumor angiogenesis, and infiltrate surrounding tissues and blood vessels . MMPs arAccording to the results of this study, there is a mean difference of (+28%) in the serum levels of MMP-9 between OSCC and healthy controls. This was in accordance with previous research by Shpitzer et al. who found that MMP-9 increased in OSCC patients by 35% to 39% compared to controls .We found no significant association between the age and gender of OSCC patients with serum MMP-9 levels. This was in accordance with previous studies examining serum levels of MMPs in breast ductal carcinoma and salivary gland tumor, with no association between age and gender . HoweverWe observed that an increase in serum level of MMP-9 significantly correlated with the increase in T grade and lymph node involvement. Similar to our findings, some previous studies have shown a significant correlation between MMP-9 with lymph node involvement . PreviouMMP-9 has an important role in ECM remodeling, metastasis, angiogenesis, apoptosis, and cancer progression . It has Hence, the MMP-9 enzyme and its inhibitors may be essential targets for anticancer therapies. Potential anticancer effects have been related to the selective inhibition of certain enzymes at specific sites . DependiLimitationIn this study, we measured the concentration of human MMP-9 in both the control and OSCC samples using a commercially available human MMP-9 sandwich ELISA kit procured from RayBioTech Human MMP-9 ELISA kit  as per manufacturer’s instructions. Since we have measured MMP-9 level in 40 patients (n=40), more number of samples are required to find the exact cut-off value for the same, and studies are under progress.However, the concentration of MMP-9 in both the control and OSCC patient samples was within the standard range of 8.23 pg/mL-6000 pg/mL and the concentration of MMP-9 in all the patient samples ranged from 136-073 pg/ml.Although only serum expression was examined in our study, future investigations of MMP-9 levels in salivary samples could be performed. The MMP-9 levels in serum with their correlation to patients' survival and response to therapy will provide valuable information.In conclusion, patients with OSCC had significantly higher serum levels of MMP-9 than healthy patients. MMP-9 could be a superior marker for determining lymph node involvement and tumor grade. Within the limits of this clinical study, serum MMP-9 has a predictive role in the diagnosis of OSCC. This study evaluated the association between MMP-9 serum levels and tumor metastasis, staging, and nodal involvement. This will provide insight into the function of MMP-9 as a significant adjuvant serological biomarker in predicting disease progression. These findings contribute to the essential involvement of MMP-9 in carcinogenesis and its potential value in aiding surgeons identify high-risk individuals who may need more aggressive therapy and close monitoring.


The study included 681 patients, of whom 97 (14.2%), 189 (27.7%), 192 (28.2%), 143 (21.0%), 47 (6.9%), and 13 (1.9%) had BACES scores of 0 to 5. Those with greater BACES scores were more likely to experience dyspnea, body weight loss, and anorexia. With severe disease, the risk of disease progression increased while the rate of treatment culture conversion decreased. After adjustment of comorbidities, higher BACES score was independently associated with the risk of mortality from respiratory causes or infection. As a simple and efficient bedside tool for assessing the severity of NTM-PD, the BACES score has the potential to be utilized as a surrogate marker for uniform severity assessment.A uniform prognostic marker is needed for nontuberculous mycobacterial pulmonary disease (NTM-PD) due to the diverse clinical course. We aimed to seek the utility of the BACES score, originally derived to predict all-cause mortality, for various outcomes. To calculate the BACES score, one point was given for each of the following factors: body mass index < 18.5 kg/m NTM can cause chronic progressive lung disease known as a non-tuberculous mycobacterial pulmonary disease (NTM-PD)3. The prevalence of NTM-PD is increasing worldwide6, and its overall mortality has been emphasized in several recent studies9. A previous study conducted in the United States reported that the 5-year mortality rate of NTM-PD was 25.0%, even without any comorbidities, which was higher than a rate of 5.7% in the general population9.Nontuberculous mycobacteria (NTM) are ubiquitous, opportunistic pathogenic bacteria in the environment, such as soil and water10; the remaining patients may experience an aggravation of the disease, leading to respiratory failure and death11. It is difficult to predict the clinical course upon diagnosis; earlier treatment is considered in those with positive smear results or cavitary lesions, whereas “watchful waiting” recommended as an alternative to immediate treatment in other patients12.NTM-PD has a broad range of clinical courses. Spontaneous negative culture conversion can occur in approximately 40% of patients with non-cavitary bronchiectasis13. The scoring system was named the BACES score; it was developed and validated in two different centers in South Korea and shown to predict all-cause mortality with excellent performance13. The BACES score was also associated with the culture conversion rate after treatment in the same population14. The BACES score needs to be validated in a different patient population to widen its use16. In this study, we aimed to discuss various utilities of the BACES score in other groups of patients with NTM-PD considering multiple clinical aspects.Recently, a simple numerical model was developed comprising five clinical variables: body mass index (BMI), age, presence of cavity, elevated erythrocyte sedimentation rate (ESR), and sex17. We excluded patients who had previously received antibiotic therapy for NTM-PD, patients with insufficient data regarding the BACES score, or who were not followed up after diagnosis.Patients diagnosed with NTM-PD at Seoul National University Bundang Hospital between May 1, 2003, and April 30, 2018, were included in this cohort study. Patients’ data were reviewed to determine whether the diagnostic criteria for NTM-PD were met18. In contrast to the previous studies19, all NTM species, including Mycobacterium avium complex (MAC), Mycobacterium abscessus complex (MABC), and Mycobacterium kansasii, were included. When a different NTM species, not the one detected initially, was seen at least twice during follow-up, the infection was considered a mixed infection20.Patient demographics, smoking history, symptoms, comorbidities, and chest computed tomography (CT) findings were retrieved from the electronic medical records. The date of NTM-PD diagnosis was defined as the date of the second culture-positive sputum collection or the date of culture-positive bronchoscopy specimen collectionThe Institutional Review Board of Seoul National University Bundang Hospital (B-2203–743-102) approved this study and waived the requirement for informed consent due to the use of anonymized data and the study's retrospective nature. This study was conducted according to the principles of the Declaration of Helsinki.2, age ≥ 65 years, presence of a cavity on chest CT, elevated ESR , and male sex13. Chest CT findings and ESR within a year of diagnosis of NTM-PD but before treatment of the disease were used. Each variable was assigned one score, with a minimum score of 0 and a maximum score of 5. The estimated probabilities of 5-year mortality according to the BACES scores are reported as 1.2% for a score of 0, 3.2% for a score of 1, 8.4% for a score of 2, 21.3% for a score of 3, 47.8% for a score of 4, and 82.9% for a score of 513. The patients were divided into three groups as follows: the mild (BACES score 0–1), moderate (BACES score 2–3), and severe (BACES score 4–5) groups14.The BACES score is composed of five variables: BMI < 18.5 kg/m23. Owing to the retrospective nature of the study, there was no uniform protocol for treating NTM-PDs, and attending physicians referred to the most recent American Thoracic Society/The Infectious Diseases Society of America/British Thoracic Society guidelines for appropriate treatment of the identified NTM species17. Culture conversion was defined as at least three consecutive negative sputum cultures after treatment. The day of collection of the first culture-negative sputum was considered the day of negative culture conversion24. The date and leading cause of death were collected from death certificates described by the attending physicians and confirmed using the Statistics Korea database using the Korean Standard Classification of Diseases, 7th edition, until December 31, 2020. Because there is no solid definition of NTM-PD-associated mortality8, two investigators (HJK and JHL) examined the causes of death associated with the respiratory system or infection were selected. Any discrepancies were resolved through group discussions. Details of the causes of death are listed in Supplementary Table Progression of NTM-PD was defined as the initiation of treatment for the diseasep-values were calculated using the chi-square or Fisher’s exact test. In contrast, continuous variables are expressed as median (interquartile range [IQR]), and p values were calculated using the Kruskal–Wallis test. For survival analyses, Kaplan–Meier curves were drawn, and p values were calculated using the log-rank test with Bonferroni adjustment for multiple comparisons. A Cox regression analysis was performed to calculate the hazard ratio (HR) with 95% confidence intervals (CIs). The p values of < 0.05 were considered statistically significant. Our observed data were utilized to calculate the 5-year probability of all-cause mortality using the Kaplan–Meier estimator.Categorical variables are expressed as numbers (percentiles) and 2. The most prevalent comorbidity was a history of tuberculosis (31.1%), followed by hypertension (24.4%), malignancy (17.3%), and diabetes mellitus (13.2%). Most of the patients had a mild-to-moderate severity of NTM-PD; 97 (14.2%), 189 (27.7%), 192 (28.2%), 143 (21.0%), 47 (6.9%), and 13 (1.9%) patients had BACES scores of 0, 1, 2, 3, 4, and 5, respectively . A history of tuberculosis and chronic pulmonary comorbidities, such as chronic obstructive pulmonary disease, asthma, and idiopathic pulmonary fibrosis, were more common in patients with severe NTM-PD than in others. Compared to patients with moderate (71.6%) and mild (61.9%) NTM-PD, patients with severe NTM-PD were more likely to be diagnosed using two repetitive sputum cultures (83.3%) rather than invasive methods (p = 0.001 by chi-square test). Patients with severe NTM-PD also had a higher initial sputum smear positivity rate  (Table Patients with severe NTM-PD were more likely to have a smoking history 51.6%) than those with mild (11.2%) and moderate NTM-PD (34.1%) (.6% than p < 0.001 by chi-square test). Symptoms of body weight loss  and anorexia  were also more common in patients with higher BACES scores (Supplementary Table Common symptoms were cough (36.0%), sputum (29.1%), hemoptysis (18.2%), and dyspnea (13.8%). Patients with severe NTM-PD were more likely to have dyspnea (26.7%) than those with mild (8.4%) and moderate (16.1%) NTM-PD (Mycobacterium kansasii (2.5%). Fifty-five (8.1%) patients were diagnosed with mixed infections. Patients with higher BACES scores had were more likely to have M. intracellulare and M. abscessus infections. In contrast, those with lower BACES scores were more likely to have M. avium and M. massiliense infections (p = 0.008 by chi-square test) , followed by MABC (14.5%) and p < 0.001). Compared to patients with mild NTM-PD, patients with moderate  or severe  NTM-PD had a higher risk of disease progression (log-rank test with Bonferroni correction p < 0.001)  had progressive disease. The median time to progression was 2.6  months, which was shorter for patients with severe NTM-PD  than for those with moderate (2.5 months) or mild (3.8 months) NTM-PD (p = 0.042 by chi-square test). However, the time to culture conversion did not reveal a significant difference between the groups (log-rank test with Bonferroni correction p = 0.596)  had culture conversion. The culture conversion rate was 65.5% in mild NTM-PD, 52.4% in moderate NTM-PD, and 40.7% in severe NTM-PD (p < 0.001)  patients had died after a median of 8.6  years following NTM-PD diagnosis. Compared to the mild group, the moderate  and severe  groups had higher risks of all-cause mortality in this separate cohort (log-rank test with Bonferroni correction 1) Table . Elderlyp = 0.038)   and severe  NTM-PD were independently associated with the risk of mortality from respiratory causes or infection. Among the comorbidities, chronic liver disease , tuberculosis , chronic obstructive pulmonary disease , and idiopathic pulmonary fibrosis  were also independently associated with a higher risk  compared to those in a similar previous study 26. Older age is a risk factor for all-cause mortality30. The presence of cavities and elevated inflammatory markers are associated with a higher risk of radiographic deterioration, all-cause mortality, and MAC-specific mortality29. Men are more likely to die with NTM-PD than women27.Our results demonstrate that the BACES score is an integrated score comprising core prognostic components, which have been identified repeatedly in earlier studies. A lower BMI is associated with a higher prevalence of NTM-PD and a higher risk of all-cause and MAC-specific mortality13. In our study, the BACES score was also associated with a higher probability of disease progression, defined as the initiation of treatment. Although recent guidelines recommend initiating therapy for cavitary disease and positive smear results12, there is no definite standard to begin treatment, and shared decision-making is critical15. An objective measure of disease severity can be helpful in such circumstances, and the BACES score can be a valid parameter. In addition, the BACES score was associated with the probability of culture conversion after treatment, which was also observed in the original derivation cohort14.Initially, the BACES score was used to predict all-cause mortality in patients with NTM-PD9. However, given the high prevalence of comorbidities, it was challenging to identify the NTM-PD-specific deaths. As indicated in our study, patients with severe NTM-PD have a higher prevalence of underlying chronic lung diseases such as asthma, interstitial pulmonary fibrosis, and chronic obstructive pulmonary disease, which are essential factors influencing NTM-PD prognosis30. Although such chronic diseases affect survival, the BACES score is an independent predictor of mortality from respiratory causes or infection. This increases the scientific power of the BACES score as a prognostic marker in NTM-PD, regardless of the underlying medical conditions.We confirmed the association between the BACES score and mortality from respiratory causes or infection, even after adjustment for comorbidities. Patients are more likely to die “of” NTM-PD rather than “with” NTM-PD as the prevalence of NTM-PD increases14. Our study may be underpowered, considering that only 256 patients were included in the survival analysis of culture conversion, compared to approximately 700 patients in the original cohort14. Nonetheless, our results indicate that mortality from respiratory causes or infection can be reduced while eradicating NTM. We suggest that achieving culture conversion is essential regardless of the time to culture conversion. Taken together, patients with a higher BACES score had a higher risk of disease progression following treatment initiation, while the culture conversion rate was lower. Given the association between the achievement of culture conversion and better survival, those with a higher BACES score are less likely to survive.Negative culture conversion was associated with fewer deaths from respiratory causes or infections but not all-cause mortality. This differs from a previous study in which negative culture conversion observed within 6 to 12 months was associated with lower all-cause mortalityMycobacterium kansasii-PD, which is comparable with the findings from previous reports33. Thirdly, while the observed mortality rate was higher than that of the derivation cohort13, the proportion of patients with subjective symptoms was lower than that reported in a recent study34. This may be due to the older age of our patients compared to the original cohort (median 66 vs. 60 years) and because we did not use a detailed questionnaire-based symptom assessment, the attending physician may not have noticed mild or intermittent symptoms like cough, sputum, or dyspnea.This study has a few limitations. First, this study was conducted in South Korea, where the BACES score was developed. Further validation studies in other geographic areas and ethnicities are required. Second, due to the study’s retrospective design, the treatment protocols could not be predefined. However, the overall rate of negative culture conversion was 54.4% in MAC-PD, 51.7% in MABC-PD, and 87.5% in 12, those with a higher BACES score can receive aggressive treatment including intravenous antibiotics, rather than “watchful waiting.” Further studies are needed to determine whether aggressive therapy for patients with a higher BACES score is associated with better outcomes.The BACES score is a simple integrated system associated with various clinical aspects. The BACES score might be a potential marker for disease progression, treatment response, death associated with the respiratory system or infection, and all-cause mortality. In addition to the recent recommendation of initiating treatment in patients with positive acid-bacilli smears and/or cavitary diseasesSupplementary Information.